Frequently Asked Questions

Selection depends on:

• Target antigens (surface vs intracellular)
• Species reactivity of the antibody
• Fluorophore compatibility with your cytometer’s laser and detector configuration
• Antigen expression level - use bright fluorochromes (PE, APC) for low-expression markers and dimmer ones (FITC, PerCP) for highly expressed antigens
• Panel complexity – minimizing spectral overlap

Online panel design tools can help you choose compatible fluorochrome and antibody combinations.

Essential controls include:

• Unstained control:Determines baseline autofluorescence.
• Single-color controls: For compensation setup.
• Fluorescence minus one (FMO) controls: Define gating boundaries for low-expression markers.
• Isotype controls: Assess nonspecific binding.

Titration ensures the optimal antibody concentration - enough to saturate target epitopes without increasing background signal. Using too much antibody leads to non-specific binding; too little may underrepresent true expression. Typically, serial dilutions (starting from 1-2 µg/test down to 0.03 µg/test) are performed to find the concentration yielding the best signal- to-noise ratio. Calculate the staining index (SI = [Mean Positive - Mean Negative] / 2×SD Negative) to identify the optimal concentration.

Surface staining targets antigens expressed on the plasma membrane (e.g., CD3, CD19), while intracellular staining targets internal proteins (e.g., cytokines, transcription factors). Intracellular staining requires cell fixation and permeabilization to allow antibody access to intracellular targets. Choosing the optimal method for fixation and permeabilization depends on the target antigen and the antibodies used.

• Use Fc receptor block (e.g., anti-CD16/CD32) before staining.
• Include proper washing steps.
• Use staining buffer with BSA or serum to block non-specific interactions.
• Optimize antibody concentrations and incubation times.
• Include viability dyes to exclude dead cells.
• Centrifuge antibodies before use (10,000×g, 5 min) to remove aggregates.

Yes, multicolor flow cytometry allows this, but you must:

• Select fluorophores with minimal spectral overlap.
• Perform proper compensation.
• Use bright fluorochromes for dim markers.
• Keep spillover spreading in mind during panel design.

Tandem dyes (e.g., PE/Cyanine7, APC/Cyanine7) are complexes of two fluorochromes coupled together for increased spectral diversity. A tandem dye works by using two covalently linked fluorophores—a donor and an acceptor—to generate a single fluorescent signal with the excitation properties of the donor and the emission properties of the acceptor. When excited by a light source, the donor fluorophore transfers its energy to the acceptor via Förster Resonance Energy Transfer (FRET), causing the acceptor to emit light at a longer wavelength. Tandem dyes are sensitive to light and fixation, and can break down into parent fluorochromes, causing compensation issues. Always protect them from light and prepare fresh samples when possible. In some cases, with certain cell types such as monocytes, tandem dyes may also exhibit non-specific binding.

• Store at 4°C in the dark.
• Avoid freezing, unless specified.
• Do not store in cytotoxic preservatives or buffers not recommended by the manufacturer. Most antibodies contain 0.09% sodium azide as preservative.
• Check manufacturer expiration dates; performance may decline over time. Tandem dyes typically have shorter expiration dates than single fluorophore conjugates.

Check the following:

• Antibody quality: Verify clone specificity and expiration.
• Antigen expression/modulation: Confirm if your cell type of interest is known to express the antigen. Some antigens are internalized after stimulation (e.g., CD62L).
• Permeabilization: Ensure correct buffer for intracellular targets.
• Instrument settings: Adjust PMT voltages, verify laser alignment, and compensation.

Direct staining uses a single fluorochrome-conjugated antibody that binds directly to the target antigen. It's faster, requires fewer steps, and reduces background noise, but offers less signal amplification.

Indirect staining uses an unlabeled primary antibody followed by a fluorochrome-conjugated secondary antibody. This method amplifies the signal (multiple secondary antibodies can bind to one primary), is more cost-effective when screening multiple targets, but takes longer and may increase background staining.

Isotype controls are antibodies of the same immunoglobulin class and fluorochrome conjugation as your test antibody but with irrelevant specificity (no known binding to your cells). They help identify:

• Non-specific Fc receptor binding
• Non-specific antibody binding
• Background fluorescence from the fluorochrome

When to use: Essential when working with new antibodies, cell types with high Fc receptor expression (macrophages, B cells), or when validating staining protocols. For well-established panels, Fluorescence Minus One (FMO) controls are often more informative.

FMO controls contain all antibodies in your panel except one, allowing you to accurately set gates by showing the spillover from all other fluorochromes into that channel. They're superior to isotype controls for:

• Multicolor panels (>4 colors)
• Setting accurate positive/negative gates
• Identifying spillover-related false positives
• Rare population analysis

Best practice: Create FMOs for markers with dim expression, rare populations, or channels with significant spillover.

Spillover occurs when a fluorochrome's emission spectrum overlaps with adjacent detectors, causing signal "bleed" into multiple channels.

Compensation is the mathematical correction that subtracts this spillover to accurately measure true fluorescence in each channel.

How to compensate:

1. Prepare single-stained compensation controls (beads or cells)
2. Use the same fluorochrome-antibody conjugates as your experimental samples
3. Run controls on the cytometer to create a compensation matrix
4. Apply the matrix to experimental data
5. Verify compensation accuracy by checking that single-positive populations don't appear in other channels

Pro tip: Use compensation beads for antibodies to avoid biological variability, but ensure bead brightness matches your cell staining intensity.

Strategic planning:

1. Prioritize markers by expression level: Assign brightest fluorochromes to dimmest antigens
2. Minimize spillover: Place markers unlikely to co-express on fluorochromes with high spillover
3. Laser configuration: Balance fluorochromes across lasers to avoid detector overload
4. Spreading: Use fluorochromes with distinct emission peaks when possible
5. Backbone markers: Place lineage/phenotyping markers on stable, bright fluorochromes

Panel design workflow:

• Start with your biological question and required markers
• Map available lasers and detectors
• Use panel design tools (e.g., Fluorescence Spectra Viewers, Panel Builders)
• Build incrementally: validate 2-3 color combinations before expanding
• Include appropriate controls (FMOs, single stains, unstained)
• Test and optimize before running precious samples

Common pitfall: Avoid putting two markers that are both highly expressed on the same cell type on fluorochromes with significant spillover (e.g., FITC and PE on T cells expressing both CD4 and CD8).